Freeze-drying is the sublimation of a frozen, aqueous solution, usually at moderate temperatures and decreased pressure for water-based solutions and has been used in many areas, including the food industry and pharmacology, and also has some history in materials research. In particular, freeze-drying has been used for conserving reagents in the field of diagnostics such as clinical chemistry or immunodiagnostics, and is particularly useful for long-term conservation of unstable fluid reagents like, for example, reference reagents, controls or calibrators for use in clinical chemistry analyzers or immunological analyzers such as protein solutions, human or animal blood sera.
Thus, the process of freeze-drying (also called lyophilization process) consists of the step of freezing the aqueous solution and the subsequent step of sublimating the frozen substance (ice crystals) in a drying process. Most of the conventional freeze-drying systems consist of equipment performing both the steps of freezing and drying in a single chamber, mainly because of cost reasons. As the freezing process in such a single chamber takes a relative long time, instable product may be shock-frozen in front of the drying/sublimation process. However, since these systems typically perform freeze-drying simultaneously on a plurality of receptacles, e.g. hundreds or even in large scale production up to 100.000 receptacles collected in, for example, a batch, such single chamber solutions have the inherent disadvantage that a high number of receptacles must be sequentially filled and added to the batch before the transfer into the freeze-dryer chamber can follow. Consequently, during the filling of the batch, a large number of filled receptacles must wait at ambient conditions until all receptacles of a batch for lyophilization have been filled, before the transfer into the chamber may be performed. During this storage, the filled receptacles, waiting in the batch, may suffer due to their instability in liquid state.
A remedy to this problem is to reduce the waiting time of the filled receptacles by having the freezing of the aqueous solution take place in a separate freezer, i.e. independently from the drying of the frozen solution. By this way, the aqueous solution, e. g. of reagents, can immediately after the respective receptacle has been filled be frozen sequentially or block-wise, e.g. using trays, and are then collected in a batch as frozen substance receptacles. Consequently, the frozen substance receptacles are not subject to deteriorating effects and can be effectively stored during the batch filling process in a frozen condition, e. g. for many hours or even longer. When the batch has been filled, a batch-wise sublimation is performed in a dryer, representing a separate entity from the freezer, without requiring that the freezing process must be stalled while preparing the next batch of frozen substance receptacles.
Conventionally, the freezing of the receptacles is performed batch-wise in a freeze dryer, by arranging the respective receptacles on pre-cooled shelfs inside the freeze dryer chamber. This has the above discussed disadvantage of deteriorating effects taking place while the batches are being prepared prior to freezing, and a number of such cooling chambers may need to be operated in parallel to avoid the deteriorating effects.
Moreover, the transport of batches into the chamber requires the chamber to be opened to replace batches for freezing. In this transition, because a large number of receptacles, with the internally product at room temperature (RT), carry a significant amount of heat into the chamber and the temperature distribution on the cooling surfaces is impacted significantly, which consequently may result in non-uniform freezing results.
This effect may be reduced by applying very low temperature cooling surfaces, but when applying very low cooling surface temperatures, e. g. below about −60° C., the receptacles may start suffering or even breaking in the freezing process.
Alternatively, instead of pre-cooling the cooling surfaces, the batch of receptacles may be arranged on non pre-cooled cooling surfaces, which are then gradually cooled to the desired temperature. However, gradually cooling the receptacles results in freezing products of another quality, for example because the freezing process is influenced by convection and supercooling effects in the aqueous solutions, which results often in an amorphous frozen product. Such products would have poor reconstitution capabilities after freeze-drying.
It is also possible to use shock-freezers to apply shock freezing to the aqueous solutions followed by storing the frozen substances in a cold chamber, e.g. at temperatures of about −45° C. By this way, the frozen substances can be stored for hours or even days, without risking any damage to the product. However, shock freezing typically results in an unpredictable outcome of the freezing process which reflects itself in non-uniform characteristics in the frozen products. Moreover, when applying shock freezing using conventional methods, the cooling effect applied to each of the receptacles varies significantly depending on the position and surrounding of the receptacle inside the batch, which contributes further to the non-uniform characteristics in the frozen products.
The book “Freeze-Drying/Lyophilization of Pharmaceutical and Biological Products, second ed. 2002, Louis Rey, Joan C. May, page 247” describes how in the conventional approach the first stage of freeze-drying involves freezing the solution to remove solvent (typ. water) from the drug and excipients through the formation of ice. The resulting semi-frozen system is cooled further to transform all components into a frozen state. A selected time/temperature profile is achieved by placing the solution, which is commonly held in glass vials or syringes or the like, onto cooled shelves. Suspended impurities in the solution or imperfections in the walls of the container initiate heterogeneous nucleation during freezing. This event almost always involves supercooling whereupon crystallization occurs below the equilibrium freezing point of the solution.
Consequently, when freezing does occur, crystal growth tends to be rapid and results in a complex mixture of crystalline, amorphous and metastable materials. In other words, during freezing, the thermal gradient within the solution is determined implicitly by the temperature of the chamber or cooled shelves, the type of receptacle used and the thermal conductivities of the materials within the solution, and is not explicitly controllable. Moreover, during the freezing process, convection effects induce material flow in the solution, which results in an even less uniform freezing environment within the solution.
In the prior art, this problem is related to as supercooling and heterogeneous nucleation, i.e. the sudden transformation from the aqueous solution to an irregular mixture of crystalline, amorphous and metastable materials, and is known to be difficult to solve. In this respect, there are for example suggestions to add salt or organic solvents to the aqueous solution, in order to influence the structure and size of the resulting ice crystals.
Alternatively, and as applied in accordance with the present invention, the negative effect of supercooling can be avoided by constraining the freezing process to take place at the melting point (equilibrium freezing point), whereby the freezing at the melting point gradually shifts the freezing front throughout the material, and therewith automatically assures constant and uniform freezing conditions throughout the freezing process. Due to the uniform freezing conditions, and the relatively slow propagation speed of the freezing front throughout the material, the freezing process taking place at the melting point is characterized by high molecular mobility and sufficient settling time for allowing the molecules to optimally arrange themselves, i.e. in line with the ice crystal structure.
The document “The Importance of Freezing on Lyophilization Cycle Development” Patapoff et al., BioPharm, March 2002, P. 16-21 discusses how directional freezing can be used to obtain straight, vertical channels in the frozen substance. For this purpose, solutions in receptacles are pre-cooled, nucleated at the bottom of the receptacle with dry-ice, and frozen in a freezing process on a −50° C. shelf, ice crystals propagate vertically and generate channels in the frozen substance, which is advantageous when water vapor needs to be drawn from the material during sublimation. It follows, for example, that the vertical freezing results in a higher sublimation rate and lower product temperature during primary drying.
Surprisingly it has been found that one of the main factors controlling growth of these channels is the temperature gradient between the bottom of the receptacle immersed in refrigerant on a conductive plate, and the medium surrounding the upper surface of the material. The temperature gradient is the driving force for ice crystal growth, and causes the ice crystals to form in a direction parallel to the heat flow, and perpendicular to the cooling surface.
On one hand a sufficiently low temperature of the conducting plate induces a stable temperature gradient for primary and secondary nucleation and driving the ice crystal growth at the melting point, while an insufficiently low temperature of the conductive plate, before primary and secondary nucleation can take place, could allow convection currents in the solution to weaken the temperature gradient, i.e. change the way of freezing and disturb and deteriorate the crystal growth at the gradually shifting melting front. However, on the other hand when very low temperatures are applied by the conductive plate, the material of the receptacle containing the liquid substance was assumed to suffer because of the low temperature applied during the complete freezing process. Consequently, having very low temperature applied during the complete freezing process may damage or even break the receptacle and thereby render the frozen sample unusable.
As to the prior art, reference is made also to J A Searles, J F Carpenter, T W Randolph; Journal of Pharmaceutical Sciences Vol 90, No. 7, July 2011, cf. page 861.
U.S. Pat. No. 4,531,373 refers to the difficulty to achieve the optimal cooling rate in directional cooling of cells, which minimizes both problems associated with cooling too slowly and the mechanical glass transition damage associated with cooling too fast. In U.S. Pat. No. 4,531,373 the focus is on the careful cooling/freezing living cells which later on can be defrosted without losing their viability.
There is thus a need for, and it would be advantageous to have to be performed on an industrial scale, a method and apparatus for directional freezing or freeze-drying of a liquid substance and providing a frozen product with improved uniformity while reducing the risk of thermally breaking or damaging the receptacles containing the liquid substances to be frozen. In the case of freeze-drying, it would also be advantageous to have the crystal structure of the frozen products construed to support the sublimation in the drying process and to reduce the reconstitution time of the freeze-dryed products (e.g. of precious pharmaceutical products).